The spliceosome is a huge complex of macromolecules, including over 200 proteins and 6 ribonucleic acids (RNA) or ribo-nucleotide chains. It catalyzes a spectacular reaction removing extraneous instructions from the blueprints of almost every protein in the human cell by extracting and splicing expanses of messenger RNA (mRNA). This function is vital as mis-splicing results in various disease states. Surprisingly, a minimized spliceosome-only two RNA pieces-can perform the catalytic step. However, this model spliceosome, or ribozyme, demonstrates a slow rate and small yields. This problem is almost certainly centered in RNA's flexibility and its tendency to adopt non-functional structures in the absence of chaperoning proteins. Previous data suggest nucleotide mimics or specifically, conformationally restricted nucleotides (CRNs), can shift non- functional conformations to properly folded/active structures when appropriately placed within an RNA. Thus, if a sample of ribozymes experience a substantially enhanced rate and yield upon CRN substitution this change would indicate an increase in the number of properly folded RNAs. This proposal aspires to design a new catalytic RNA that better recapitulates splicing's two-step reaction by using these nucleotide mimics that are restricted to conformations common in properly folded RNAs. This strategy may simulate the presence of proteins by enforcing the RNA conformation found in the spliceosome's catalytic complex. This proposal details three aims targeted at identifying key positions for CRN inclusion in a newly designed RNA-only spliceosome, herein termed the splicezyme, as well as recounting the benefits of rational CRN incorporation for RNA structural biology studies. Aim 1 (proposal section C1) describes the thought processes resulting in the new splicezyme construct, and discusses the characterization of the basal activity of this ribozyme prior to CRN substitution. Initial reported findings contained therein suggest the splicezyme successfully generated the first-step product. Proposal section C2 chronicles the structural and biochemical data used to identify candidate positions for rational CRN substitution. Here, the recently-determined structure of a closely related ribozyme defines nucleotides that adopt the same conformation as CRN. Also, there are data implying this configuration in other nucleotide positions. Aim 2 describes splicezyme CRN-substitution positions extrapolated from these data. Finally, aim 3 (proposal section C3) proposes to characterize the three-dimensional structure of an undetermined portion of the splicezyme using CRNs and NMR spectroscopy, a technique that enables spatial measurements with atomic resolution. This aim hypothesizes that CRNs will reduce misfolded RNA accumulation, a major problem of RNA structural biology. In devising the splicezyme and developing CRNs as probes, this research seeks insight into RNA structural aspects involved in splicing catalysis and the spliceosome's intermolecular interactions, which will provide means for rational manipulation in various future biomedical and therapeutic pursuits. PUBLIC HEALTH RELEVANCE: As many as 95% of all human genes are spliced-have extraneous genetic information removed, often in a tissue-specific manner-by the spliceosome. Thus, malfunctions in this ubiquitous process can produce a variety of disparate disease states that are otherwise unrelated, e.g. hemachromatosis, dementia, and blindness. Therefore, characterizing the fundamental attributes of the catalytic spliceosome complex through methods described here will provide insight into conditions affecting various body systems and introduce a foundation for future biomedical and therapeutic pursuits.